Process for the purification of a red fruit extract containing anthocyanosides, extract obtained from the process and use of said extract

ABSTRACT

Process for purifying a red fruit extract containing anthocyanosides, according to which:  
     said extract is taken up in an aqueous solution;  
     the aqueous extract is cooled until it reaches a homogeneous temperature of less than 15° C.;  
     after filtering the aqueous extract, the permeate obtained is recovered and loaded onto a macrocrosslinked polymeric resin;  
     the resin is then rinsed with demineralized water;  
     then the resin obtained is eluted with an alcoholic eluting solution;  
     finally, the eluate obtained is concentrated and then dried.

[0001] The invention relates to a process for purifying a red fruitextract containing anthocyanosides. It also relates to the extractobtained by said process and its use.

[0002] In the remainder of the description and in the claims, “redfruit” denotes in particular elderberry (Sambucus nigra), blackcurrant(Ribes nigrum), redcurrant (Ribes rubrum), mulberry (Morus nigra), grape(Vitis vinifera), and more particularly bilberry (Vaccinium myrtillusand other species of the genus Vaccinium), this being withoutlimitation.

[0003] In general, the term “anthocyanosides” denotes a heterosaccharideresulting from the combination of an anthocyanic genin with one or moreglycosidic groups. These anthocyanosides exist in the form of monomers,but also of dimers, oligomers and polymers according to an increasingdegree of polymerization. The anthocyanoside-anthocyanoside bond occursbetween the C4 of the C nucleus and the C8 of the A nucleus.

[0004] In the present case, anthocyanosides denote the structures in theform of monomers whose molecular weight varies from 400 to 600 Daltons.Certain forms of dimers may also be present whose molecular weight maybe up to about 1000 Daltons.

[0005] Red fruits are rich in pigments called anthocyanosides which havebeen demonstrated to have an effect on blood microcirculation by actingin particular as vitamin P factor, as antioxidant, as plateletaggregation inhibitor and as anti-free radical agent. These propertieshave been recognized for the treatment of disorders involving retinalcirculation, in the treatment of functional disorders of hairbrittleness and visual disorders of vascular origin. These propertieshave also been demonstrated for the treatment of hesperanopia andmyopia, in the proprietary medicinal products marketed under the trademarks DIFRAREL® 100 and DIFRAREL® E by LEURQUIN MEDIOLANUM laboratories.

[0006] Several processes for extracting anthocyanosides from plants orfrom plant portions have been proposed.

[0007] Document FR-A-2 299 385 thus describes a process for extractinganthocyanins from grape marc comprising an extraction step proper,followed by a step for concentrating the extract obtained.

[0008] According to this process, the extraction step consists intreating the marcs with an acidic aqueous extraction solution (pH=2)supplemented with SO₂ in the hot state (between 40 and 55° C.). Theclear solution obtained, containing the anthocyanosides, but also theacids, salts, polyphenols and proteins, is then concentrated. To dothis, it is loaded onto a resin. The resin is then eluted with aneluting solution containing either a ketone, an amide or an aqueoussolution of an alkali or alkaline-earth metal hydroxide. Theanthocyanins are finally separated from the eluate obtained.

[0009] It is evident from the examples described, in particular example1, that starting with an extract containing 120 mg of anthocyanins perliter, an extract is obtained at the outlet of the resin which comprises800 mg per liter of anthocyanins, that is to say an extract concentrated6.6 fold.

[0010] However, even if the process used makes it possible to markedlyincrease the concentration of anthocyanosides in the extract, thisconcentration is not at all indicative of a purification of theanthocyanosides.

[0011] Furthermore and in particular, the initial extraction of theanthocyanosides with a solvent supplemented with SO₂ leads to theattachment of the anthocyanosides to the resin in a modified form, whichis therefore capable of disrupting the physicochemical characteristicsof the anthocyanoside and therefore its activity.

[0012] More recently, a process for extracting and then purifyinganthocyanosides from bilberries was proposed in the document EP-A-412300.

[0013] The extraction step proper consists in placing frozen fruits incontact with an aqueous solution of methanol, each extraction extendingover a period of four hours.

[0014] The extract obtained is then purified. To do this, it is firstconcentrated under vacuum, the resulting concentrate then beingsupplemented with sodium bisulfite. A bond is then formed between theanthocyanosides and the bisulfite ions. After stirring for three hoursand neutralization by adding a sodium hydroxide solution, the extractobtained is loaded onto a column of a nonionogenic polymer resin andthen the column is eluted with purified water. The eluate is thenacidified to pH=1 with concentrated hydrochloric acid (HCl). To removethe SO₂, nitrogen is then bubbled through the solution obtained so as todissociate the anthocyanosides-bisulfite complex. This dissociationleads to the release of sulfur dioxide. The aqueous solution is thenextracted with butanol. The butanolic solution is supplemented with 14volumes of ethyl acetate. After allowing to stand overnight, theprecipitate is dried at 40° C.

[0015] As above, the process used still requires the use of bisulfiteions leading to the formation of an anthocyanoside-bisulfite complexcapable of altering the physicochemical properties of the desiredanthocyanosides.

[0016] Furthermore, the use of nitrogen for regenerating theanthocyanosides results in emissions of sulfur dioxide which can createenvironmental problems and problems of cumbersome treatments of thedischarges.

[0017] It is finally also important to emphasize the multitude ofsolvents used (methanol, butanol and ethyl acetate) and the cumbersomechemical treatments which this extraction process involves: use of SO₂,NaOH, acidification to pH=1 with concentrated HCl and use of nitrogen.

[0018] A process for extracting coloring substances and moreparticularly a process for extracting anthocyanosides from the berriesof fruits, bilberries, blackcurrants or cranberries requiring inparticular the use of a weakly polar supercritical solvent which issubsequently distilled off is known from patent FR 2641983. Theextraction residue is purified by the customary physical means, inparticular by chromatography on a polyamide column and then elution withhydrochloric methanol.

[0019] The anthocyanosides thus obtained and in particular those ofbilberry are active ingredients of medicaments.

[0020] From this prior state of the art, the invention relates to aprocess for purifying an extract of red fruits containinganthocyanosides capable of solving the following problems:

[0021] dispensing with the use of any SO₂-type additive which is likelyto modify the structure and the physicochemical characteristics of theanthocyanosides extracted;

[0022] avoiding any emission of harmful substances and in particular ofsulfur dioxide;

[0023] increasing the concentration of anthocyanosides in the purifiedextract obtained;

[0024] carrying out a purification without structural modification ofthe anthocyanosides;

[0025] reducing as much as possible the quantity of residual solventscontained in the purified extract;

[0026] obtaining an extract in which the residual solvents are clearlyidentified, and the low quantity of which complies with the ICH(International Conference on Harmonization of Technical Requirements forRegistration of Pharmaceuticals for Human Use) guidelines.

[0027] To do this, the subject of the invention is a process forpurifying a red fruit extract containing anthocyanosides, according towhich:

[0028] said extract is taken up in an aqueous solution;

[0029] the aqueous extract is cooled until it reaches a homogeneoustemperature of less than 15° C.;

[0030] after filtering the aqueous extract, the permeate obtained isrecovered and loaded onto a macrocrosslinked polymeric resin;

[0031] the resin is then rinsed with demineralized water;

[0032] then the resin obtained is eluted with an alcoholic elutingsolution;

[0033] finally, the eluate obtained is concentrated and then dried.

[0034] In this way, by dispensing with any solvent supplemented withSO₂, the process of purification of the invention, carried out undergentle conditions, causes no modification of the native physicochemicalcharacteristics, and therefore no denaturation of the anthocyanosidesextracted. This characteristic may be confirmed by the superposition ofthe HPLC chromatographic profiles as a function of the retention timefor the extract, on the one hand, and for the starting red fruit, on theother hand.

[0035] Furthermore, it is observed that the combination of various stepsof the process, and in particular the cooling step followed by thefiltration and passage over a resin, leads, quite surprisingly, to theextracted anthocyanoside titer in the final dry extract beingconsiderably increased. For a temperature of the aqueous extract greaterthan 15° C., sufficient purification of the anthocyanosides is notobtained.

[0036] In a first embodiment of the invention, the process ofpurification is carried out on an alcoholic red fruit extract obtainedaccording to the following process:

[0037] the pulp is first of all separated from the whole red fruits;

[0038] said pulp is then brought into contact with an alcoholicextraction solution;

[0039] then the solid phase is separated from the liquid phase;

[0040] finally, the major portion of the residual alcohol contained inthe liquid phase is evaporated under vacuum so as to obtain an alcoholicconcentrate.

[0041] Advantageously, the solvent used for the alcoholic extraction ischosen from the group comprising methanol, ethanol, butanol and acetone.

[0042] In practice, the alcoholic extraction is carried out at roomtemperature in at least two successive steps, each lasting 20 minutes.The solvent is then evaporated off. In addition, it is also possible toenvisage carrying out the extraction of the anthocyanosides not from thepulp alone, but from whole fruits.

[0043] Furthermore, the solid phase/liquid phase separation may becarried out by any known means, in particular centrifugation.

[0044] According to a second embodiment of the invention, the process ofpurification is carried out starting with extracts of red fruits whichare commercially available or with prepurified anthocyanoside extract,each provided in liquid or powdered form. In this case, the fruitextract or the prepurified extract may then be taken up, before thepurification step, either with alcohol, in particular methanol, or withwater.

[0045] In the process of purification of the invention, the cooling ofthe red fruit extract is advantageously carried out until thetemperature of said extract is homogeneous and less than 10° C.,preferably less than 5° C., this temperature being maintained for atleast twelve hours.

[0046] As regards the step of filtration of the aqueous extract, it maybe carried out on a cellulose filter or a stainless steel gauze with acut-off of between 0 and 100 micrometers or equivalent.

[0047] As already stated, the permeate obtained from the filtration stepis then loaded onto an adsorbent macrocrosslinked resin.

[0048] Moreover, in order to further increase the titer and theconcentration of anthocyanosides in the final extract, the alcoholicsolution with which the anthocyanosides are eluted from the resin ispreferably an aqueous solution of ethanol whose ethanol concentration isbetween 10 and 90%, advantageously close to 40%.

[0049] The eluate obtained is concentrated at a controlled temperaturein the region of 30° C. and then freeze-dried or spray-dried so as toobtain a powder.

[0050] It is moreover observed that the powder obtained contains a verysmall residual quantity of alcoholic solvent, of less than 3000 ppm formethanol (if the purification is carried out from a methanolic extract)and less than 2000 ppm for ethanol. These residual solvents areidentified by GC and are the only ones contained in the final powder.

[0051] The invention also relates to the extract which may be obtainedby the process of purification described above.

[0052] In addition, by virtue of its high titer of originalanthrocyanosides and its low content of residual solvents, the extractmay be used in any cosmetic, pharmaceutical or dietetic composition. Itmay also be used in nutraceutical compositions, functional foods,enriched foods and dietary supplements.

[0053] The invention and the advantageous which result therefrom willemerge more clearly from the following examples of implementation, insupport of the appended figures in which:

[0054]FIG. 1 is a comparison of an HPLC chromatogram of the finalextract obtained according to example 1 relative to that of the originalfruit and that of the original pulp;

[0055]FIG. 2 is an identical comparison carried out starting with theextract obtained according to example 2.

[0056] The two examples which follow relate to the extraction ofanthocyanosides from bilberry pulp.

EXAMPLE 1 1/Separation of the Pulp

[0057] 125 kilos of bilberries from Lithuania are pressed by any knownmeans. After pressing, the pulp representing 52.24 kilos and the juicerepresenting 71.48 kilos are separated.

2/Extraction

[0058] Two successive extractions are then carried out starting with 35kilos of bilberry pulp with each time 210 liters of methanol (that is 1volume of pulp per 6 volumes of extraction solution) for twenty minutesat room temperature. At the end of the extraction, the pulp is pressedso as to recover the methanolic fraction. The methanol is thenevaporated off under vacuum, the temperature of the extract then beingequal to 22° C.

3/Purification

[0059] The concentrate is then taken up in 70 liters of demineralizedwater (that is one volume of pulp per 2 volumes of water).

[0060] The aqueous extract thus obtained is then stored at a temperatureof less than 15° C. overnight.

[0061] The solution is then filtered on a 50 micrometer stainless steelgauze.

[0062] A macrocrosslinked resin, for example the resin XAD761 marketedby ROHM & HAAS, is then loaded with the aqueous extract. 25 liters ofresin are used in practice.

[0063] Once the resin has been loaded, it is rinsed with 105 liters ofdemineralized water (that is one volume of pulp per 3 volumes ofdemineralized water).

[0064] The resin is then eluted with 200 liters of ethanol at 40%.

[0065] The eluate is then concentrated under vacuum, the temperature ofthe extract then being between 26 and 31° C.

[0066] To obtain a powder, the eluate is then spray-dried.

[0067] The anthocyanoside content of the final extract is equal to50.46% of the final weight, that is 53.50% by weight of dry matter.

[0068] These results are expressed relative to cyanidin chloride, astandard used for HPLC analysis, reference 0909S, supplier:Extrasynthèse.

[0069] It is observed that the residual quantity of solvent in the finalextract is:

[0070] 0 ppm for butanol,

[0071] less than 3000 ppm for methanol,

[0072] less than 2000 ppm for ethanol.

[0073] The results of a chromatogram produced by HPLC of the finalextract (1) relative to that of the original fruit (2) and pulp (3) arepresented in FIG. 1.

[0074] As shown in this chromatogram, the profile for the extractedanthocyanosides is identical to that for the native anthocyanosidespresent in the original pulp or fruit, thus indicating that thephysicochemical characteristics of the anthocyanosides are not adverselyaltered throughout the extraction and purification process.

[0075] It is therefore possible to think that the extract obtained,whose anthocyanoside composition is identical to that present in theoriginal fruit, retains all the recognized qualities of the fruit.

EXAMPLE 2

[0076] Example 1 is reproduced starting with a second batch ofbilberries from Poland.

[0077] It is observed that the anthocyanoside concentration obtained isequal to 49.1% by weight of the final extract, that is 52.01% by weightof dry matter.

[0078] The residual quantity of solvents is:

[0079] 0 ppm for butanol,

[0080] less than 3000 ppm for methanol,

[0081] less than 2000 ppm for ethanol.

[0082] As above, FIG. 2 represents the HPLC chromatogram for the pulpfor the fruit and for the extract obtained.

[0083] It is observed, in an identical manner, that the profile for theanthocyanosides contained in the extract (4) is identical to thatcontained in the pulp (5) and the fruit (6).

COMPARATIVE EXAMPLE 3

[0084] In this example, a comparison is made of various characteristicsof commercial bilberry extract, respectively:

[0085] a bilberry extract marketed by the company INDENA calledANTHOCYANOSIDE;

[0086] and a second bilberry extract marketed by the company VINYALScalled BILBERRY DRY EXTRACT (25% anthocyanosidine);

[0087] relative to an extract produced according to the process of theinvention.

[0088] The criteria selected are:

[0089] the anthocyanoside concentration calculated by HPLC relative to acyanidin chloride standard;

[0090] the residual quantity of solvent in the final extract.

[0091] The results are presented in the following table. INDENA VINYALSINVENTION SPECIFICATION SPECIFICATION SPECIFICATION INVENTION RESULTAnthocyanosides 23.75-26.25% 23.75-26.25% 45-55% 51.80% Residual ≦30,000ppm ≦30,000 ppm ≦3000 ppm 1035 ppm organic solvents ethanol ≦30,000 ppm? ≦2000 ppm 890 ppm methanol   ≦300 ppm ?  ≦300 ppm  145 ppm othersolvents    ≦50 ppm ?   ≦1 ppm  ≦1 ppm Chromatographic not not originalsuperposition profile determined determined fruit of the two profiles

[0092] As shown in the table, the extract obtained according to theprocess of the invention makes it possible to obtain a very highanthocyanoside concentration compared with the commercial extract.

[0093] A very low residual quantity of organic solvent is also observed.

[0094] Finally, while nothing is indicated in the specifications for theINDENA and VINYALS extracts as regards the chromatographic profile, itis observed that the chromatographic profile for the extract of theinvention may be superposed on that for the original fruit.

[0095] The advantages of the invention as regards the restitution in theextract of the native anthocyanosides of fruits are clearly evident fromthe description.

[0096] The use of conventional extraction solvents, which are removedthroughout the process such that residual solvents are only present intrace amounts in the extract obtained, will be noted in particular.

[0097] Likewise, the process does not require the use of SO₂-typeadjuvants to the extraction solvents or of chemical compounds, whoseremoval is highly delicate and poses a number of environmental problems.

[0098] Moreover, it is observed that the anthocyanosides are notdenatured throughout the process and preserve a composition identical tothat of the original fruit, thus making it possible to preserve theirentire activity.

[0099] Finally, the anthocyanoside concentration of the extractobtained, which is in the region of 50% by weight of dry matter, shouldbe emphasized.

1. Process for purifying a red fruit extract containing anthocyanosides,according to which: said extract is taken up in an aqueous solution; theaqueous extract is cooled until it reaches a homogeneous temperature ofless than 15° C.; after filtering the aqueous extract, the permeateobtained is recovered and loaded onto a macrocrosslinked polymericresin; the resin is then rinsed with demineralized water; then the resinobtained is eluted with an alcoholic eluting solution; finally, theeluate obtained is concentrated and then dried.
 2. Process according toclaim 1, characterized in that the aqueous extract is cooled until itreaches a homogeneous temperature of less than 10° C.
 3. Processaccording to claim 1, characterized in that the red fruit extract onwhich the purification is carried out is an alcoholic extract obtainedaccording to the following steps: the pulp is first of all separatedfrom the whole red fruits; said pulp is then brought into contact withan alcoholic extraction solution; then the solid phase is separated fromthe liquid phase; finally, the major portion of the residual alcoholcontained in the liquid phase is evaporated under vacuum so as to obtainan alcoholic concentrate.
 4. Process according to claim 3, characterizedin that the solvent used during the alcoholic extraction is chosen fromthe group comprising methanol, ethanol, butanol and acetone.
 5. Processaccording to either of claims 1 and 2, characterized in that thepurification is carried out on a prepurified extract provided inpowdered form or in liquid form.
 6. Process according to one of thepreceding claims, characterized in that the alcoholic eluting solutionis an aqueous solution of ethanol whose ethanol concentration is between10 and 90%.
 7. Process according to claim 5, characterized in that thealcoholic eluting solution is an aqueous solution of ethanol whoseethanol concentration is equal to 40%.
 8. Purified extract which may beobtained according to the process which is the subject of claims 1 to 7.9. Use of the extract of claim 8 for the preparation of apharmaceutical, cosmetic, dietetic or nutraceutical composition, infunctional foods, in enriched foods and dietary supplements.